Real-Time Monitoring of ATG8 Lipidation in vitro Using Fluorescence Spectroscopy

Autophagy is an essential catabolic pathway used to sequester and engulf cytosolic substrates via a unique double-membrane structure, called an autophagosome. The ubiquitin-like ATG8 proteins play an important role in mediating autophagosome membrane expansion. They are covalently conjugated to phosphatidylethanolamine (PE) on the autophagosomes via a ubiquitin-like conjugation system called ATG8 lipidation. In vitro reconstitution of ATG8 lipidation with synthetic liposomes has been previously established and used widely to characterise the function of the E1 ATG7, the E2 ATG3, and the E3 complex ATG12–ATG5-ATG16L1. However, there is still a lack of a tool to provide kinetic measurements of this enzymatic reaction. In this protocol, we describe a real-time lipidation assay using NBD-labelled ATG8. This real-time assay can distinguish the formation of ATG8 intermediates (ATG7~ATG8 and/or ATG3~ATG8) and, finally, ATG8-PE conjugation. It allows kinetic characterisation of the activity of ATG7, ATG3, and the E3 complex during ATG8 lipidation. Furthermore, this protocol can be adapted to characterise the upstream regulators that may affect protein activity in ATG8 lipidation reaction with a kinetic readout. Key features • Preparation of ATG7 E1 from insect cells (Sf9 cells). • Preparation of ATG3 E2 from bacteria (E. coli). • Preparation of LC3B S3C from bacteria (E. coli). • Preparation of liposomes to monitor the kinetics of ATG8 lipidation in a real-time manner.

This protocol is used in: eLife (2023), DOI: 10.7554/eLife.89185 Autophagy is an essential catabolic pathway used to sequester and engulf cytosolic substrates via a unique double-membrane structure, called an autophagosome.The ubiquitin-like ATG8 proteins play an important role in mediating autophagosome membrane expansion.They are covalently conjugated to phosphatidylethanolamine (PE) on the autophagosomes via a ubiquitin-like conjugation system called ATG8 lipidation.In vitro reconstitution of ATG8 lipidation with synthetic liposomes has been previously established and used widely to characterise the function of the E1 ATG7, the E2 ATG3, and the E3 complex ATG12-ATG5-ATG16L1.However, there is still a lack of a tool to provide kinetic measurements of this enzymatic reaction.In this protocol, we describe a real-time lipidation assay using NBD-labelled ATG8.This real-time assay can distinguish the formation of ATG8 intermediates (ATG7~ATG8 and/or ATG3~ATG8) and, finally, ATG8-PE conjugation.It allows kinetic characterisation of the activity of ATG7, ATG3, and the E3 complex during ATG8 lipidation.Furthermore, this protocol can be adapted to characterise the upstream regulators that may affect protein activity in ATG8 lipidation reaction with a kinetic readout.

Background
Autophagy is a well-conserved bulk degradation pathway from yeast to mammals.It occurs ubiquitously in response to metabolic demands and plays an important role in maintaining cellular homeostasis and cell survival (Mizushima and Komatsu, 2011).Upon autophagy induction, cytosolic materials are sequestered and enclosed by a doublemembrane structure, called an autophagosome, leading to the degradation of the content after fusion with lysosomes.One of the key discoveries in autophagy field is ATG8 lipidation via the ubiquitin-like conjugation systems (Mizushima, 2020; Nishimura and Tooze, 2020).The ubiquitin-like ATG8 is first primed by a cysteine protease ATG4 to expose its C-terminal glycine.Then, the activated ATG8 is conjugated to ATG7 (E1) in an ATP-dependent manner and transferred to ATG3 (E2).It is finally covalently conjugated to the headgroup of phosphatidylethanolamine (PE), catalysed by ATG12-ATG5-ATG16L1 complex (hereafter, the E3 complex).Lipidated ATG8 is commonly used as autophagosomal membrane marker, and ATG8 lipidation is monitored to assess the autophagy activity in cells (Mizushima et al., 2010).In vitro reconstitution of ATG8 lipidation with synthetic membrane models, such as large unilamellar vesicles (LUVs), is also well established (reviewed in Huang et al., 2022).The end-point level of lipidated ATG8, which is usually examined by SDS-PAGE, has been used to assess the function of ATG7 (Taherbhoy et al., 2011;Noda et al., 2011), ATG3 (Nath et al., 2014), and the E3 complex (Lystad et al., 2019).However, this end-point readout cannot track ATG8 conjugation reactions in a real-time manner or provide more information on the reaction rate.Recently, we designed a real-time lipidation assay using human ATG8 proteins (LC3B/GABARAP) N-terminally labelled with 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) (Zhang et al., 2023).As NBD fluoresces brightly around 535 nm when it is located in a hydrophobic environment, we use it to dynamically track the hydrophobic environment that ATG8 N-terminus encounters during the lipidation reaction (provided by the ATG7 or ATG3 interfaces and/or the membrane).Interestingly, the increased NBD signals in the real-time assay, in addition to responding to membrane environments, also reflect the enzymatic activity of ATG7, ATG3, and the E3 complex, which can be readily adapted to evaluate the function of ATG7, ATG3, the E3 complex, or other upstream regulators, and provide a kinetic measurement in future studies.This protocol provides the setup for the real-time lipidation assay using NBD-labelled LC3B, as an example.there is no excess dye (unreacted dye migrates in dye front).7. Mix μL of glycerol with 500 μL of NBD-labelled LC3B S3C to a final concentration of 20%.Aliquot the protein, flash freeze in liquid N2, and store at -80 °C .

E. Liposome preparation
To prepare liposome stock (1 mL, 2 mM final concentration) 1. Mix POPC and DOPE lipids at a ratio of 50:50 (% mol).To characterise the effect of each component on the real-time assay, add the amount of buffer instead of the component of interest.For example, for "no ATG7" condition, prepare the reaction mix without ATG7 and add buffer instead.The protein stock can be diluted to a lower concentration so that it will be easier to pipette a larger volume of protein.

2 .
Dry the lipids under nitrogen gas for 5 min and further dry in the Eppendorf TM Concentrator for 2 h. 3. Add 1 mL of assay buffer onto the lipid film and vortex thoroughly until the lipid film is fully resuspended.4. Conduct five freeze-thaw cycles in liquid nitrogen and 42 °C water bath until the lipid solution is fully thawed.5. Prepare the unilamellar vesicles with the Mini Extruder.Extrude the liposome solution first by passing the solution 21 times through a 0.2 μm membrane.Then, extrude the solution by passing it at least 41 times through a 0.1 μm membrane.Note: It is recommended to extrude the liposomes through a 0.2 μm membrane before a 0.1 μm membrane in order to make homogeneous liposomes.6. Check the liposome size by Zetasizer Nano ZS [for example, Figure 1: average size 105.5 nm, PDI (polydispersity index) = 0.071].If the size of liposomes is not homogeneous (PDI > 0.2), repeat the extrusion step through the 0.1 μm membrane.

Table 1 . Components for the real-time ATG8 lipidation assay (total volume: 78.4 μL) Component Final concentration Volume to add (stock concentration)
2. Table1indicates the concentration and volume for each component in the real-time lipidation ATG8 assay.